3D-localization microscopy and tracking of FoF1-ATP synthases in living bacteria

TL;DR

This study employs advanced super-resolution microscopy techniques to observe and analyze the spatial distribution and diffusion behavior of FoF1-ATP synthases in living E. coli bacteria under physiological conditions.

Contribution

It introduces a novel application of PALM and SIM microscopy combined with a sliding observation window for quantitative diffusion analysis in bacterial membranes.

Findings

FoF1-ATP synthases are spatially distributed in bacterial membranes.

The diffusion coefficient along the cell's long axis was quantitatively measured.

The method accounts for membrane curvature in diffusion analysis.

Abstract

FoF1-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single FoF1-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of the observation area in the membrane with its significant membrane curvature has to be considered. Therefore, we applied a 'sliding observation window' approach (M. Renz et al., Proc. SPIE 8225, 2012) and obtained the one-dimensional diffusion coefficient of FoF1-ATP synthase diffusing on the long axis in living E. coli cells.